Multi-slide assembly including slide, frame and strip cap, and methods thereof

ABSTRACT

A multi-slide assembly includes various components that alone or in combination facilitate testing of test samples with minimal manipulation of assay components. Moreover, the various device components permit centrifugation, culturing and analysis of each test sample to be performed in the same assembly. A frame retains a plurality of reaction vessel assemblies between a pair of opposing channels that are configured to slidably receive the opposing ends of each reaction vessel assembly. A strip cap seals the openings in each reaction vessel using cap members having sealing rings circumscribing the external walls thereof to form compression seals with internal walls of the reaction vessels. Furthermore, in a method of making a multi-well slide assembly, an ultrasonic welding process removably bonds a plurality of wells to a slide plate to provide an adequate seal therebetween during centrifugation, yet still enable separation thereof by an operator.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.10/139,550, filed on May 6, 2002 by Bunn et al. (issued as U.S. Pat. No.6,803,239), which is a divisional application of U.S. Ser. No.09/510,717, filed on Feb. 22, 2000 by Bunn et al. (issued as U.S. Pat.No. 6,383,820), which is a divisional application of U.S. Ser. No.08/958,521 filed on Oct. 27, 1997 by Bunn et al., (issued as U.S. Pat.No. 6,096,562) all entitled “MULTI-SLIDE ASSEMBLY INCLUDING SLIDE, FRAMEAND STRIP CAP, AND METHODS THEREOF,” which applications are incorporatedby reference herein.

FIELD OF THE INVENTION

The invention is generally directed to cell culturing, immunological andmolecular tools for use in biomedical research and diagnosticsapplications. More specifically, the invention is directed to cellculture, immunological and molecular tools such as slides, wells,vessels and the like for use in research or diagnostic testing ofbiological test samples.

BACKGROUND OF THE INVENTION

Cell culture vessels such as flasks, dishes, slides, wells, culturetubes and the like are utilized in a number of biomedical diagnosticsapplications to test for the presence of microorganisms in test samplessuch as patient specimens from humans or other animals. Likewise, othertypes of reaction vessels similar in format but differing in surfacecharacteristics to cell culture vessels are utilized for immunological,molecular and biochemical analysis of the same.

For example, microorganisms may be tested for to assist in the diagnosisof an infection. Some infectious diseases are distinctive enough to beidentified clinically. Most pathogens, however, can cause a widespectrum of clinical symptoms in humans, many of which are not unique toa particular pathogen. Therefore, it is often necessary to usemicrobiologic laboratory methods to identify the specific organism thatis causing a disease.

One method used to detect the presence of a microorganism causing aninfection is to isolate and culture in an artificial medium themicroorganism causing the infection. Both the presence and number ofmicroorganisms in a patient's specimen can assist in defining the causeof a disease. To use this process, a specimen from a patient is placedin a liquid, solid or semi-solid medium that permits the growth ofselective microorganisms. The medium may also include inhibitorysubstances that prevent the growth of microorganisms in the medium,other than the microorganisms the maker of the medium has selected. Ifthe microorganism is present in the patient's specimen, it will grow inthe medium and its growth will be detected.

Another method that may be used to identify the presence of amicroorganism is to expose a living cell line that is known to besusceptible to particular microorganisms to a test sample from apatient. If the microorganism is present in the patient's specimen, thecells may exhibit cytopathic effects induced by that microorganism,which may be detected and confirmed by a fluorescent labeled monoclonalantibody. Typically, with such a process a cell line is grown on asuitable surface such as a cover slip disposed in the Petri dish, dramvial, culture tube or other vessel. Alternatively, the cell culture maybe grown directly on the surface of a suitable vessel. The cell cultureis inoculated with a clinical test sample, and then some of thesevessels may be centrifuged to quickly introduce the microorganism intothe cells. Typically, the cell culture is then incubated for a period oftime to grow the organism, whereby the presence or non-presence of themicroorganism is later analyzed, typically through visual inspectionusing an optical reader such as a microscope.

In many cases, to grow or culture cells both a solid surface and aliquid medium are needed. The solid surface provides a location uponwhich the cells can adhere, and the solid support typically mimics thecell's natural environment in the tissue from which they were derived.Often, the flat surfaces of tissue-culture flasks, trays, Petri dishes,multi-well culture plates, and even the inside surfaces of large rollerbottles make ideal support surfaces for growing cells.

The medium in which the cells are immersed is the cells' source ofnutrients. It is an artificial environment similar enough to the cells'natural environment to permit their continued growth and proliferation.The basic formulas of culture media typically consist of water, saltsand amino acids, to which supplements such as serum, antibiotics, orgrowth factors can be added.

Most cell culture vessels are typically not well suited for performingall tasks in a diagnostic process. For example, a culture tube or Petridish is often suitable for inoculation and/or incubation; however, manysuch cell culture vessels are not well suited for centrifugation and/oranalysis. In general, cell growth occurs best on a modified plasticsurface, while analysis and other processing is best performed on aglass surface. Consequently, test samples often must be transportedbetween various vessels during the diagnostic process. Moreover, withvessels such as dram vials, culture tubes or Petri dishes, each testsample must be handled individually, which can become cumbersome whenworking with numerous samples.

Multi-well culture plates may also be used in diagnostic testing. Amulti-well plate has multiple wells formed into a two-dimensional arraywithin which one or more cell lines are grown. Often, however, suchmulti-well plates are restrictive in that it is difficult to culturemultiple cell lines in a single plate due to different culture timesrequired for each type of cell line. Moreover, there is a possibility ofcross-contamination between wells. Also, if one cell culture in amulti-well plate becomes contaminated or otherwise inoperative,typically the entire plate must be discarded.

It has also been found that centrifugation causes a number of concernswith many conventional cell culture vessels. Typically, such vesselsmust be relatively sturdy to withstand the forces that occur incentrifugation. Moreover, an adequate seal must be maintained duringcentrifugation to prevent loss of the cell culture and/or contaminationof other cultures, or the laboratory environment.

Accordingly, it is desirable to maintain a tight seal with various cellculture vessels subjected to centrifugation. However, a tight seal on avessel may induce an aerosol effect when the sealing member for thevessel is removed. It should be appreciated that whenever a sealingmember is removed under a tight seal, a vacuum is temporarily induced inthe vessel. When the vacuum is released after the sealing member isfully removed, viruses or other biological agents or contaminants may bereleased into the atmosphere due to the sudden pressure change in thevessel. Such agents may be dangerous to operators, and they may alsocause contamination of nearby samples.

Another concern is the vaporization of certain harmful processingchemicals such methanol, ethanol, isopropanol, dimethyl suphoxide(DMSO), phenol, or chloroform. Consequently, great care must be taken inremoving a sealing member from conventional cell culture vessels.

Another conventional cell culture vessel is a multi-well slide assemblysuch as the SonicSeal four well slide available from Nalge NuncInternational, which includes multiple wells joined together and securedto a slide plate through a breakable ultrasonic weld. An opener may beused to remove the upper structure of the wells from the slide platesuch that the cell cultures disposed on the slide plate may be analyzed.However, such assemblies are not designed for centrifugation and includeno suitable manner of sealing each well during centrifugation. Theloose-fitting lid provided with such assemblies is insufficient totightly seal each well.

Such assemblies are typically welded together using a two-stepultrasonic welding process. The upper structure is provided with a thinflange with a triangular cross-section on a mating end thereof (commonlyreferred to as an energy director) that is melted during ultrasonicwelding to weld the upper structure to the slide plate. In the firststep, the upper structure is ultrasonically welded to a distance ofapproximately one half of the length of the energy director to energizethe molecules therein. Then, in the second step, a stronger weld isformed between the upper structure and slide plate through limitedadditional ultrasonic welding that further energizes the molecules butdoes not cause the energy director to become completely fused—therebyproviding the break-apart property of the slide. After bonding, about 88percent of the energy director is used up, with the mating surfacebetween the upper structure and slide being only about 0.015 incheswide, which is only about 88 percent of the width of the base of theenergy director, and only about 34 percent of the thickness of thesidewalls of the upper structure. The resulting bond is air- andliquid-proof and has sufficiently high mechanical strength for culturingpurposes. However, the bond tends to leak during and/or after exposureto centrifugation forces, and thus is not suited for use in acentrifuge.

Therefore, a significant need exists for improved tools and an enhancedmanner of performing cell culturing, diagnosis and/or testing ofbiological test samples with greater efficiency, reliability andaccuracy. Specifically, a need exists for tools that are particularlysuited for multiple activities to minimize the effort, time andpotential contamination concerns associated with transferring testsamples, cell cultures and the like between vessels.

SUMMARY OF THE INVENTION

The invention addresses these and other problems associated with theprior art in providing a multi-slide assembly including variouscomponents that alone or in combination facilitate testing ofbiological, chemical or molecular test samples with minimal manipulationof assay components. Specifically, various operations such ascentrifugation, inoculation, culturing and/or analysis of a test samplemay be performed in the same reaction vessel to speed up testing and/orto minimize cross-contamination between multiple samples. Batchprocessing of numerous test samples may therefore be performedexpediently and reliably.

Consistent with one aspect of the invention, a method is provided fortesting a test sample in which a reaction vessel defined at leastpartially by a sidewall member removably coupled to a slide plate isinoculated with a test sample and centrifuged. In addition, the sidewallmember is separated from the slide plate to permit analysis of the testsample on the slide plate.

Consistent with another aspect of the invention, a frame is used toretain a plurality of reaction vessels, with each reaction vesselincluding opposing ends. The frame includes a pair of opposing channelsincluding first and second ends and configured to slidably receive theopposing ends of each reaction vessel, and first and second retainingmechanisms respectively disposed proximate the first and second ends ofthe pair of opposing channels and configured to retain each reactionvessel within the pair of opposing channels.

Consistent with yet another aspect of the invention, a method isprovided for performing a common operation on a plurality of testsamples, each test sample being housed in one of a plurality of reactionvessels, each vessel of the type including opposing ends. The methodincludes placing the plurality of reaction vessels in a frame includinga pair of opposing channels including first and second ends and slidablyreceiving the opposing ends of each reaction vessel, and first andsecond retaining mechanisms respectively disposed proximate the firstand second ends of the pair of opposing channels and retaining eachvessel within the pair of opposing channels. The method also includesperforming the common operation on the plurality of test samples whilethe plurality of reaction vessels are retained in the frame.

Consistent with another aspect of the invention, a strip cap is alsoprovided for use in sealing an opening in the reaction vessel having aninternal wall defining the same. The strip cap includes a cap memberhaving an external wall, a mating portion of which is configured to abutthe internal wall of the vessel around a perimeter of the opening, and asealing ring circumscribing the external wall of the cap member andconfigured to form a compression seal with the internal wall of thevessel.

Consistent with a further aspect of the invention, a multi-well slide isprovided including a slide plate, a plurality of wells coupled to theslide plate, each including an internal wall defining an opening to thewell, and a strip cap. The strip cap includes a strip member, aplurality of cap members coupled to the strip member, each cap memberhaving an external wall, a mating portion of which is configured to abutthe internal wall of one of the plurality of wells around the perimeterof the opening thereto, and a plurality of sealing rings, each sealingring circumscribing the external wall of one of the plurality of capmembers, and each sealing ring configured to form a compression sealwith the internal wall of one of the plurality of wells.

Consistent with yet another aspect of the invention, a method of makinga reaction vessel of the type including at least one well defined by asidewall member removably coupled to a slide plate is provided,including forming a bond that is sufficient to withstand centrifugationforces between the sidewall member and the slide plate via ultrasonicwelding; and controllably weakening the bond between the sidewall memberand the slide plate via ultrasonic welding to permit selectiveseparation of the sidewall member from the slide plate.

Consistent with a further aspect of the invention, a reaction vessel isprovided, including a slide plate removably secured to a sidewall memberthrough an ultrasonically-welded and controllably-weakened bond. Thesidewall member includes an energy director disposed at an end thereof,the energy director having a base with a width that is substantiallyequal to that of the sidewall member proximate the end thereof. The bondincludes a fused junction between the slide plate and the sidewallmember that is formed of substantially the entire energy director andthat defines a mating surface that is substantially equal to the widthof the base of the energy director.

These and other advantages and features, which characterize theinvention, are set forth in the claims annexed hereto and forming afurther part hereof. However, for a better understanding of theinvention, and of the advantages and objectives attained through itsuse, reference should be made to the Drawing, and to the accompanyingdescriptive matter, in which there is described exemplary embodiments ofthe invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an exploded perspective view of a multi-slide assemblyconsistent with the invention.

FIGS. 2A and 2B respectively illustrate cross-sectional views of anupper structure and slide plate of a multi-well slide before and afterultrasonic welding.

FIGS. 3A and 3B are respectively top plan and side elevational views ofthe strip cap shown in FIG. 1, with portions thereof cut away in FIG.3B.

FIG. 4 is a cross-sectional view of one cap member in the strip cap ofFIG. 2, illustrating the seal formed with a well on a multi-well slideassembly.

FIG. 5 is a side elevational view of one cap member in the strip cap ofFIG. 2, with portions of a multi-well slide assembly cut away toillustrate removal of the strip cap from the multi-well slide assembly.

FIG. 6 is a top plan view of an alternate strip cap to that of FIG. 2.

FIG. 7 is a top plan view of the frame shown in FIG. 1.

FIG. 8 is a side cross-sectional view of the frame, taken through lines8-8 of FIG. 7.

FIG. 9 is an end cross-sectional view of the frame, taken through lines9-9 of FIG. 7.

FIGS. 10A-10C are side cross-sectional views of the frame of FIG. 6,illustrating insertion of a plurality of multi-well slides into theframe.

FIG. 11 is a top plan view of the multi-slide assembly of FIG. 1.

FIG. 12 is a bottom plan view of the multi-slide assembly of FIG. 1.

FIG. 13 is a top plan view of an alternate multi-slide assemblyconsistent with the invention.

FIG. 14 is a side elevational view of the opener shown in FIG. 1.

FIG. 15 is an end elevational view of the opener shown in FIG. 1.

DETAILED DESCRIPTION

Turning to the Drawing, wherein like numbers denote like partsthroughout the several views, FIG. 1 is an exploded perspective view ofa multi-slide assembly 10 consistent with the principles of theinvention. Assembly 10 generally includes a plurality of multi-wellslide assemblies 20, each including a multi-well slide 30 and a stripcap 50. Each assembly 20 may also include a lid 25 that may be usedduring incubation to cover each multi-well slide when its associatedstrip cap is removed, thus minimizing potential contamination withmicroorganisms, dust, etc.

The plurality of multi-well slide assemblies 20 are housed in a frame100 that is suitable for performing batch operations on the cellcultures within the multi-well slide assemblies. Also shown is a pry-baropener 200 that may be used to separate the upper structures of themulti-well slides from the slide plates thereof.

The discussion hereinafter will focus on cell culture applications ofthe invention. However, it should be appreciated that the invention maybe utilized in other applications, e.g., in immunological and molecularapplications, cell-based assay applications, etc. Consequently, whilevarious components of the embodiments described hereinafter, such asvessels, assemblies, surfaces, and the like, may be described withreference to cell culturing applications, one skilled in the art willappreciate that the principles of the invention may be applied to otherapplications of reaction vessels and reaction vessel assemblies ingeneral without departing from the spirit and scope of the invention.

Multi-Well Slide Assemblies

Each multi-well slide 30 shown in FIG. 1 includes a slide plate 32having opposing top and bottom surfaces 33 a, 33 b, opposing ends 34 a,34 b, a beveled front edge 34 c and a rear edge 34 d. Each slide plate32 also includes a frosted writing surface 35 and a raised well floor 36disposed within each well and upon which a cell culture is grown.

An upper structure 40 including a plurality of wells 42 isultrasonically welded to slide plate 32. Each well 42 forms a cellculture chamber with slide plate 32, and each includes an inner wall 42a and a top edge 42 b defining an opening 42 c in each well. Theplurality of wells 42 are integrally molded to one another and supportedby flange 44. Upper structure 40 forms a sidewall member that defineseach well in conjunction with slide plate 32. It should be appreciatedthat other sidewall member designs, e.g., defining any number of wells,may be used in the alternative. For example, a sidewall member may onlydefine one well.

The design of multi-well slide 30 is similar in some respects to the No.138121 SonicSeal slides available from Nalge Nunc International. Theslide plate has standard dimensions of a microscope slide. Moreover,either or both of the slide plate and the upper structure may be formedof Permanox® plastic or other similar materials that have suitablechemical resistance for performing culturing, analysis and otherprocessing, as well as biological compatibility for facilitating cellgrowth thereon. The upper structure 40 is ultrasonically welded to theslide plate 32 to form a liquid tight seal. Moreover, the upperstructure 40 is removable by prying the upper structure from the slideplate 32 with a pry-bar opener 200 that breaks the ultrasonic weld.

The aforementioned SonicSeal slides have been found to not beparticularly well-suited to centrifugation due to the lack of strengthin the ultrasonic welds used thereon. To address this concern, it isdesirable to provide a coupling between the upper structure 40 and slideplate 32 that is suitable of withstanding centrifugation, but whichstill enables the removal of the upper structure by a user.

Ultrasonic welding in general is known in the art. See, e.g., U.S. Pat.No. 4,715,911, which is incorporated by reference herein. Consistentwith the invention, it is desirable to utilize a two-step,weld-by-distance ultrasonic welding process that creates an initial bondin a first welding step that is sufficiently leak-proof during and aftercentrifugation. Then, in a second welding step, the upper structure andslide plate are further compressed to cause a secondary melt thatcontrollably weakens the bond without compromising the leak-proof sealestablished by the first step.

For example, as shown in FIG. 2A, a tapered energy director 48 (e.g.,with a triangular cross-section) may be provided on the end of asidewall 46 on upper structure 40. Energy director 48 may have a lengthL of about 0.014 to about 0.024 inches (0.36 to 0.61 mm), morepreferably about 0.019 inches (0.49 mm). Energy director 48 tapers atabout a 30 degree angle from a base 48 a to a tip 48 b, with a width Wthat is preferably about 0.017 inches (0.42 mm). Sidewall 46 to whichenergy director 48 is coupled preferably has a thickness of about 0.043inches (1.09 mm).

For the first weld cycle, a pressure of about 15 to about 30 psi, morepreferably about 20 psi may be used, with a weld distance of about 0.014to about 0.019 inches (0.36 to 0.48 mm), more preferably about 0.017inches (0.42 mm), and a weld duration of about 0.5 to 1.0 seconds, morepreferably about 0.8 seconds. The first weld cycle fully bonds the upperstructure to the slide plate as a result of the energy director becomingfully molten during welding.

For the second weld cycle, a pressure of about 20 to about 40 psi, morepreferably about 30 psi may be used, with a weld distance of about 0.001to about 0.005 inches (0.03 to 0.13 mm), more preferably about 0.004inches (0.09 mm), and a weld duration of about 0.5 to 1.0 seconds, morepreferably about 0.8 seconds. A dwell time of about 3 to about 10seconds may be used, more preferably about 5.0 seconds, to permit somecooling of the components while under pressure and thereby minimizefailures. Additional suitable weld parameters include a stroke positionof about 0.1 inches (2.54 mm), a mechanical stop of about 4.3 inches(109.22 mm), an end weld of about 6.0 inches (152.4 mm), a pre-triggerof about 5.5, a moderate down speed, and a trigger of about 2.0. Itshould be appreciated that the parameters may vary depending upon theparticular ultrasonic welding machine used.

It has been found that, by generating a full weld to approximately thefull length of the energy director in a first welding step, a bond isgenerated between the upper structure and slide plate that can withstandcentrifugation forces (e.g. at least 700 G's for an hour) and still beleak-proof during and after centrifugation. Then, by welding in thesecond weld at least the remaining length of the energy director at agreater pressure (typically about 10 psi greater) than in the firstweld, a controlled weakening of the bond may be generated to reduce thebreakage resistance of the bond to a level that permits separation ofthe upper structure from the slide plate, but that is still capable ofwithstanding centrifugation. In addition, the width of the bond ispermitted to occupy, in the minimum, the full width of the energydirector, and in the maximum, the full thickness of the sidewall, whichis believed to contribute to the improved robustness of the bondcompared to conventional designs. In the embodiment discussed above, thetotal weld distance can be about 0.02 inches (0.51 mm) for a energydirector with a length of about 0.019 inches (0.49 mm), and the width ofthe bond therefore can range from about 0.017 inches (0.42 mm) wide toabout 0.043 inches (1.09 mm). Therefore, as shown in FIG. 2B, theresulting bond between slide plate 32 and upper structure 40 uses up theentire energy director, and defines a mating surface 49 therebetweenthat is substantially the thickness T of sidewall 48.

In addition, it has been found to be beneficial to cool moldedcomponents prior to ultrasonic welding. For example, the components maybe allowed to air cool for 12-24 hours before welding. Alternatively, asimilar degree of cooling may be performed for two or three minutes onan air conditioned cooling conveyor that transports components from themolding process to the welding process.

Conventional welding processes that attempt to first energize thecomponents, and second weld the components together have been found tonot provide the same combination of resistance to centrifugation forcesand separability with controllable results. For example, a conventionalwelding process may utilize a two-step process, with a first steputilizing an air pressure of 10 psi, a distance of 0.010 inches, and aweld time of 1.2 seconds, and a second step utilizing an air pressure of15 psi, a distance of 0.007 inches, and a weld time of 0.5 seconds.Other weld parameters of the conventional process utilize a dwell timeof 5.0 seconds, a mechanical stop of 0.420 inches, and end weld of 6.110inches, a pretrigger of 5.726 inches a trigger of 1.6 and a moderatedown speed. Even by increasing welding pressure, time and/or distance totheoretically increase the bond strength, it has been found thatconventional processes of this type do not provide suitably consistentresults. In particular, it is believed that the width of the bond, whichis only a portion of the width of the energy director, and less of aportion of the width of the sidewall, does not provide suitablerobustness for centrifugation applications.

In other conventional welding processes, the full length of an energydirector may be used up during welding, resulting in a bond thatoccupies the full width of the sidewalls. However, the bonds resultingfrom these conventional processes are permanent and are not breakable byan operator without damage to the welded components. Consequently, theseprocesses are not suitable for many of the applications disclosed hereinthat utilize the separability of a slide plate and an upper structure toperform analysis of test samples.

It should be appreciated that the above-described welding parameters maybe varied in different applications. It should be appreciated thatmaterials other than Permanox® plastic may require different weldingcharacteristics. For example, for polystyrene, it may be desirable toutilize a longer energy director (e.g., about 0.025 to about 0.035inches, or 0.64 to 0.89 mm), with a correspondingly greater welddistance in the first weld step (e.g., about 0.022 to about 0.031inches, or 0.56 to 0.79 mm).

It should also be appreciated, however, that the design of multi-wellslide 30 is but merely one type of cell culture vessel suitable for usewith the various components of the invention. For example, variousenclosed cell culture vessels including flasks, dishes, culture tubesand structures including a plurality of wells in various geometricshapes, etc. may be used. Moreover, various non-enclosed cell culturevessels such as slides, wafers, discs, plates, microarrayed surfaces,etc. may also be used, as may other types of reaction vessels suitablefor other immunological and molecular applications, for example.Accordingly, the invention should not be limited to the particulardesign of multi-well slide 30, nor specifically to the material and/orthe manner of coupling the upper structure to the slide plate, disclosedherein.

Strip Cap

Strip cap 50 of multi-well slide assembly 20 is shown in greater detailin FIGS. 3A and 3B. In general, strip cap 50 includes a strip member 52joining a plurality of cap members 60, one of which is provided for eachwell on multi-well slide 30.

As best shown in FIG. 4, each cap member 60 includes an external wall 62having a mating (or side wall) portion 63 and bottom portion 64. Matingportion 63 is typically cylindrical in shape, or other suitable shape tomatch the well with which it is being used. For example, while each well40 in multi-well slide 30 is circular in cross-section, othercross-sectional profiles, such as square or rectangular, may also beutilized for each well, thereby necessitating an alternating shape foreach cap member. Mating portion 63 of each cap member 60 may also betapered, (e.g., at about 3°) to facilitate insertion of the cap memberinto the opening 42 c of each well 42.

Bottom portion 64 of each cap member may be flat or domed. A flatprofile provides a lower profile for the cap member, while a domedmember may be useful in smaller designs to reinforce the cap member. Anannular bevel at 64 a is defined between mating portion 63 and bottomportion 64 to further facilitate insertion of each cap member into itsassociated well.

Returning to FIG. 4, one or more sealing rings, e.g. sealing rings 66,68 are provided on each cap member to provide a compression seal againstthe inner wall 42 a of each well 42 proximate the perimeter of opening42 c. Any number of sealing rings may be utilized, however, it has beenfound that one sealing ring may not provide as strong a seal as multiplesealing rings, in part due to the redundancy provided by multiple rings.

Moreover, it has been found that the use of multiple seals can provide aventing effect when the cap member is removed, which minimizes thevacuum created in the chamber, and consequently minimizes aerosoleffects associated therewith.

For example, as shown in FIG. 5, a channel 76 is defined between sealingring 66, 68, such that as cap member 60 is removed from its associatedwell, channel 76 places well 42 in fluid communication with theatmosphere prior to complete removal of cap member 60. In particular, ascap member 60 is peeled off with a general tilting motion, channel 76will first be exposed to the atmosphere as shown at 80. As the capmember is further peeled off and tilted, sealing ring 68 disengages fromside wall 42 a, as shown at 82, to place channel 76 in fluidcommunication also with the interior of well 42. The channel istherefore capable of venting air into the well as the cap member isremoved to reduce the degree of vacuum, and therefore minimize anyaerosol effects.

Returning to FIG. 4, each cap member 60 also includes an internal wall70 having an optional reinforcement ring 72 defined along the wallbetween sealing ring 66, 68 to assist and maintain the integrity of thewall. Moreover, a plurality of transversely-oriented ribs 74 may also beprovided on the internal wall of each cap member to further addintegrity to the cap member. Ribs 74 are not required, for example asshown by strip cap 50′ of FIG. 6. Strip cap 50′ includes plurality ofcap members 60′ that includes sealing rings as well as a reinforcementring 72′ on the internal wall 70′. However, no transverse ribs areprovided on the internal wall thereof.

Strip member 52 (best shown in FIGS. 3B and 4) extends generallytransverse to the mating portions of cap member 60 to form a flange 53that overlaps the top edge 42 b of each well 42 when the strip cap issecured to a multi-well slide. Flange 53 prevents contaminates such asdust or various biological agents from resting on the edge of each well.Moreover, the flange reduces well-to-well contamination. Furthermore,flange 53 prevents the cap member from being inserted too far into thewell, and keeps the user's finger off the top edge of the well tofurther minimize contamination thereof.

A pair of tabs 54 also extend from the ends of strip member 52. Eachincludes fingernail ridges 54 a that assist in the manual removal of thestrip cap. Moreover, one of the tabs 54 may also include an orientationmarking 54 b extending transverse to ridges 54 a so that strip member 52may be secured to a multi-well slide in a repeatable, predeterminedorientation. Consequently, the risk of cross-contamination due toplacing strip member 52 on backwards relative to a previous installationof the same is reduced.

The design of strip member 52 also permits tweezers to be utilized toremove the strip cap by grasping flange 53. Moreover, if strip cap 50 isconstructed of a soft material, a knife or scalpel may be used toseparate the individual cap members such that each is separatelyremovable with tweezers. This enables wells to be opened and closedindividually. For example, this may enable a sealed control sample to bemaintained while various testing operations are performed on the testsamples. It also enables various samples to be inoculated at differentstarting times to account for the different growth rates of various celllines.

Typically, strip cap 50 is integrally injection molded with a flexiblematerial such as a USP Class 6 or compatible medical-rated plastic thatis not toxic to test samples or other biological agents that may come incontact with the strip cap during testing. For example, one suitablematerial is low-density polyethylene, although other materials, such ashigh density polyethylene or polypropylene, among others, may also beused.

The design of strip cap 50 provides significant advantages overconventional designs. For example, strip cap 50 provides a strong sealthat is suitable for centrifugation as well as transportation ofmulti-well slide assemblies. The seal provided by such a strip capenables cell cultures of living cell lines to be commercially preparedin wells, sealed within the assemblies along with a suitable transportmedium, and then sold, stored and/or transported to customerlaboratories for later use. Customers are then able to merely remove thestrip caps and inoculate the cell lines as desired to perform testing,without first having to culture their own cell lines.

Another significant advantage is that the strip caps are replaceablesuch that after inoculation, the multi-well slide assemblies may beresealed and utilized during centrifugation. This seal may assist inmaintaining the media's pH during shipment as well as during thediagnostic process.

Frame

FIG. 7 illustrates frame 100 of FIG. 1 in greater detail. The frameincludes left and rights sides 102 and 104 and front and rear ends 106and 108. A pair of opposing channels 110 are provided along sides 102,104 and a pair of retaining mechanisms 130, 150 are disposed at the ends106,108 thereof. A lower support member 160 with a plurality of accessapertures 162 defined therein supports and separates the opposingchannels at a predetermined distance from one another.

Frame 100 is typically sized at about 5.025″ by 3.365″ to provide afootprint that is substantially similar to a conventional microwellplate or multi-well cell culture dishes (e.g., 6, 12, 24, 48, 96, 384,etc. well plates). Moreover, the height of frame 100 is slightly smallerthan that of a microwell plate. The dimensions enable frame 100 to beused with the various processing and analysis equipment that currentlyexist for processing microwell plates or multi-well cell culture dishes.For example, various liquid handling systems such as washers,dispensers, robotic fingers, pipettors, and other ancillary equipmentsuch as centrifuges having centrifuge buckets/carriers have beendeveloped to utilize assemblies having such footprint. Moreover, variousoptical viewers, including microscopes (specifically, the stagesthereof), spectrophotometers, luminometers, fluorimeters, andcolorimetric readers, among others, may also be compatible with such afootprint. Other robotics, manufacturing systems and coating systems,may also recognize such footprint.

Frame 100 is typically constructed of an autoclavable, chemicallyresistant, strong, and freezable material such as Noryl plasticavailable from General Electric, which is a 20% glass-filled modifiedpolyphenylene oxide. Other materials, including various plastics andmetals, whether transparent or opaque, may also be used consistent withthe invention. The frame may or may not be reusable.

As shown in FIGS. 8 and 9, each opposing channel 110 of frame 100includes upper and lower walls 112, 114. Upper wall 112 is partitionedinto a retaining portion 112 a that extends along a longitudinal axis ofthe channel and parallel with lower wall 114. An access portion 112 b ofupper wall 112 extends at an acute angle relative to the longitudinalaxis, terminating at an open end to define access opening 140 throughwhich multi-well slide assemblies are inserted and removed from frame100. It should be appreciated that each channel may be defined by otherstructure in frame 100. For example, lower wall 114 may be defined on aseparate member, rather than being contiguous with lower support member160. In addition, should the opposing ends of the particular reactionvessel assemblies to be retained by frame 100 not be planar (as withmulti-well slide assemblies 30), alternate configurations and dimensionsof the channels may be utilized to retain suitable structure on thereaction vessel assemblies for use therewith.

Retaining mechanism 130 includes a stop member 132 and retainingmechanism 150 includes a stop member 152, each of which close off an endof both opposing channels. Moreover, retaining mechanism 130 includes adetent 134 that extends from stop member 132 towards the opposite end ofthe channel. The detent 134 is spaced from lower wall 114 approximatelythe same distance as the upper wall retaining portion 112a—approximately the width of the structure that is retained within thechannels. Detent 134 is beveled along an upper surface thereof tofacilitate movement of retained objects over the detent. Retainingmechanism 150 may also include an upper surface 154 (See FIG. 7), whichmay include integrally-molded alphanumeric information, or which mayprovide a writeable surface or space for a label such that an operatorcan identify or annotate the assembly.

It should be appreciated that various other retaining mechanisms mayalso be used to close the ends of the opposing channels and therebysecure reaction vessel assemblies within the frame. For example, eitheror both of the retaining mechanisms may be removable to selectively openor close the end of the opposing channels. The removal or retainingmechanisms may be secured by any number of means, including clips,screws, detents, tabs, etc. In general, any structure that abutsretained objects to prevent movement thereof beyond the ends of thechannels may be used. Moreover, the retaining mechanisms may abut suchobjects either within the channels or in the space defined therebetween.However, it is believed that the illustrated structure providesadvantages over various alternatives as no moving parts are required,and as the configuration thereof is relatively simple and reliable formanufacturing and use.

Frame 100 is configured to hold four multi-well slides such asmulti-well slide assemblies 20 of FIG. 1. However, frame 100 could beused to secure other reaction vessel assemblies, including variousplanar assemblies such as slides, plates, wafers, etc., whereby theplanar surfaces retained within by the channels are coplanar withsurrounding structure on the assembly. Various other assemblies,including microarrayed surfaces, microwell and multi-well plates,flasks, etc., may also be used. Moreover, the retained surfaces need notbe co-planar with the surrounding structure of the assembly nor needthey have a planar shape. In general, any reaction vessel assemblieshaving opposing ends suitable for retention within a pair of channelsmay be secured in the manner disclosed herein in frame 100.

FIGS. 10A-10C illustrate the insertion of multi-well slide assembliesinto frame 100. First, FIG. 10A illustrates the insertion of a secondmulti-well slide assembly 20 b through access opening 140 of frame 100,with a first multi-well slide assembly 20 a already retained therein. Itshould be noted that assembly 20 b is slightly angled to orient plate 32b thereof between the upper and lower walls 112, 114 of frame 100. Toinsert assembly 20 b into frame 100, the assembly is slid toward therear end of the frame, in the general direction toward retainingmechanism 150.

Next, as illustrated in FIG. 10B, the insertion of three assemblies 20a-20 c into frame 100 proceeds in much the manner described above. Toinsert a fourth assembly 20 d, however, each of the first threeassemblies 20 a-20 c must be slid rearward to abut one another as wellas abut stop member 152 of retaining mechanism 150. This provides enoughseparation within each channel 110 to permit assembly 20 d to be slidinto frame 100 a sufficient distance to be tilted downward past detent134. The spacing provided between detent 134 and stop member 152 isequal to or slightly larger than the combined widths of the fourassemblies. In the alternative, the distance between detent 134 and stopmember 152 may be slightly less than the total width of assemblies 20a-20 d such that some resistance is encountered when inserting orremoving assembly 20 d from frame 100.

Next, as illustrated in FIG. 10C, once all assemblies 20 a-20 d aredisposed between channels 110, all assemblies are slid forward towardretaining mechanism 130 to abut assembly 20 b against stop member 132,and thereby leave a small gap between assembly 20 a and stop member 152.Consequently, assemblies 20 a-20 d are secured within the channels.

The gap remaining within each channel once all assemblies 20 a-20 d areinserted therein may be slightly larger than the width of detent 134 topermit each assembly to be removed easily when slid rearward, yetretained securely when the assemblies are slid forward. In thealternative, the gap may be made slightly less than the width of thedetent, such that some resistance must be overcome to dislodge anassembly when all are retained within the frame.

As shown in FIGS. 11 and 12, frame 100 provides clear access to both thetop and bottom portions of each multi-well slide assembly 20. As shownin FIG. 11, as well as FIG. 8, a single upper access aperture 170 istypically defined in frame 100 such that free access to each ofassemblies 20 a-20 d is provided. Moreover, as shown in FIG. 12, lowersupport member 160 includes one or more access apertures 162 that permitassemblies 20 a-20 d to be viewed and accessed through the bottom of theframe (See, e.g., wells 40 a, 40 d, which are visible through clearplates 32 a, 32 d). As shown in FIG. 12, access apertures 162 may bedefined by voids formed in member 160. In the alternative, apertures 162may be formed by transparent portions of member 160. Moreover, member160 may be simply constructed of a single planar member formed withtransparent material. In these latter instances, the access apertureswould merely provide visual access apertures that permitted viewing butnot physical access.

Access aperture 162 enable viewing of cell cultures from the top orbottom of the assembly, e.g. by using an optical viewer such as amicroscope. Moreover, by configuring access apertures 162 as voids, anoptical viewer may be oriented directly proximate the slide plates ofassemblies 20 a-20 d if necessary.

In the alternative, access apertures may be omitted from frame 100,e.g., by providing a planar opaque lower support member. Such a designmay be useful, for example, to provide high contrast with the cellcultures for use with other types of optical viewers.

An additional function of member 160 is to provide support forassemblies 20 a-20 d, particularly during centrifugation. When utilizingmulti-well slide assemblies 30 having removable upper structures, bowingmight otherwise break the well seals and cause leakage and contaminationof the cell cultures during this process. The design of lower supportmember 160 therefore supports assemblies 30 to prevent any such bowing.

FIG. 13 illustrates another multi-slide assembly 300 that illustratesthe use of coordinating indicia on a plurality of multi-well slideassemblies 310 a-310 d and a frame 330. For example, each multi-wellslide assembly, e.g., multi-well slide assembly 310 a, may include astrip cap 320 in which each cap member 322 a-322 d includes an indicia324 a-324 d that assists in identifying a particular well. Typically,each indicia 324 a-324 d is one of a series of letters or numbers (e.g.,letters A-D as shown in FIG. 13) that are matched with correspondingindicia identifying each well on the associated multi-well slide (notshown in FIG. 13), so that the strip cap may be removed and replaced ina repeatable orientation. Each indicia may be integrally molded into thebottom portion of each cap member, or in the alternative, may be printedonto the surface of each cap member in any suitable manner known in theart.

Moreover, the indicia on each cap member may also coordinate withmatching indicia 334 a-334 d molded or printed onto surface 332 of frame330 to provide a visual cue that facilitates proper installation of eachmulti-well slide assembly 310 a-310 d into frame 330. Frame 330 may alsohave additional indicia, e.g., a manufacturer's logo 336, as well astransverse indicia 338 a-338 d located on surface 337. Indicia 338 a-338d typically includes a series of letters or numbers (e.g., numbers 1-4as shown in FIG. 13) to identify each multi-well slide assembly 310a-310 d when installed in frame 330. Indicia 338 a-338 d typically arelocated so that they are visible when assemblies 310 a-310 d areinstalled in the frame. Moreover, indicia 334 a-334 d and 338 a-338 dcooperate to identify each well in assembly 300 (e.g., the well in thetop left corner is identified as well A1, and the well in the bottomright corner is identified as well D4). Other indicia and manners ofidentifying wells and the like may also be used in the alternative.

Method of Use

Multi-slide assembly 10 and its various components have numerous uses inthe biomedical diagnostics applications. For example, as described ingreater detail below, one principle use for multi-slide assembliesconsistent with the invention is in diagnosing the presence of a virusin a test sample such as a patient specimen. However, it should beappreciated that this usage is but one of numerous other applicationswhere it is desirable to perform life science cell and tissue culturing,such as in research and diagnostic screening, viral screening, cancerscreening, drug screening, etc. Therefore, the invention should not belimited to the particular application described herein.

For viral diagnostic screening, a live cell line is grown in eachmulti-well slide assembly. The cell lines selected typically have aparticular sensitivity to a given virus for which is desired todetermine the presence/non-presence thereof in a test sample. The virususes the cell to replicate itself resulting in cytopathic effect (CPE)or an antigen that can be detected using a monoclonal antibody.

For example, for the influenza and parainfluenza viruses, the chosencell line may be RHMK (Rhesus Monkey Kidney) cells. For CMV(cytomegalovirus), the chosen cell line may be MRC-5 (Human Lung). ForVZV (Varicella-Zoster Virus), the cell line may be A549. For adenovirus,the cell line may be A549, or alternatively, HEp-2 (Human Larynx). Forother viruses such as enterovirus, RSV (Respiratory Syncytial Virus),HSV (Herpes Simplex Virus), and others, the cell line may be any of theRHMK, MRC-5, A549, or HEp-2. Live cell lines or cultures, which aresensitive to other viruses, may also be used in the alternative.

Cells may be grown in each multi-well slide assembly in a clinicallaboratory in a manner known in the art. In the alternative, theassemblies may be prepared by a manufacturer of such assemblies usingcommercially-grown cells.

In either case, the empty wells within each assembly are inoculated withcells and the cells are cultured in a growth medium such as Dulbecco'sModified Eagle's Medium (DMEM). When the cells are grown in the clinicallaboratory in which the diagnostic testing is to be performed, thegrowth medium may be removed such that test samples may be used toinoculate the cell culture. If, however, the assemblies are prepared bya manufacturer, the growth medium may be removed and replaced with atransport medium that keeps the cells stable during transport. Themulti-well slide assemblies are then sealed with strip caps, packagedand transported to customers. Moreover, such assemblies may be mountedin groups of four within frames for packaging and transport.

It should be appreciated that, given the flexibility provided by themulti-well slide assemblies as well as the use of multiple assemblies ina frame, any number of combinations of cell types may be provided ineach multi-slide assembly. Typically, each well in a multi-well slideassembly has the same cell type, since otherwise different culturingtimes for different cell types may make it more difficult to ensureadequate growth of cell cultures within a given assembly.

Once cell cultures are formed in the multi-well slide assemblies,selected assemblies are inoculated with test samples such as patients'specimens. Multi-well slide assemblies may be inoculated individually,or alternatively, can be placed in a frame with any desired combinationof cell cultures represented in the different assemblies.

If the multi-well assemblies are pre-manufactured assemblies suppliedwith commercially-grown cell cultures, the strip caps are first removed.Next, the transport medium may be aspirated from the wells, and thewells are optionally rinsed, e.g., with a rinse agent such as HanksBalanced Salt Solution (HBSS). In addition, a small amount (e.g. about0.02 ml) of growth/maintenance medium such as EMEM (Eagle's MinimumEssential Medium) or serum free medium, is optionally added to maintainthe cell cultures during inoculation and to buffer test sample toxicity.Typically, the amount of growth/maintenance medium is small so that anyviruses in the test sample are maintained in close proximity to the cellculture.

Next, predetermined amounts of test samples are introduced into thewells to inoculate the cell cultures. Then, the strip caps arereinstalled on each of the assemblies in preparation for centrifugation.

If the multi-well slide assemblies are not yet disposed in a frame, theymay be so mounted such that the frame may be placed in a suitablemicrowell plate carrier bucket of a centrifuge. Centrifugation is thenperformed on all the assemblies concurrently, e.g. at 700 G's for aboutone hour. Centrifugation assists in introducing any viruses into a cellculture more rapidly to speed up the diagnostic process.

Next, once the cell cultures have been centrifuged, the strip caps areremoved from the assemblies and a growth medium is optionally added. Theassemblies are then covered with lids and are incubated at an elevatedtemperature for a predetermined time (e.g., 36° Celsius for multipledays in a CO₂ incubator). The lids are utilized in lieu of the stripcaps in this process to permit some degree of CO₂ transfer duringincubation. In some applications, strip caps may instead be utilized toseal the wells from air. During incubation, it may also be desirable toperiodically replace the growth medium as necessary.

Upon completion of incubation, the test samples may be analyzed forpotential cytopathic effects or antigen expression. Typically, the lidsor strip caps are removed and any growth media is aspirated from thecell cultures. The cell cultures may be fixed, stained, etc., asnecessary to permit suitable analysis of the cell cultures. For example,the cells may be fixed with acetone, dried and then stained with a dropof appropriate monoclonal conjugate. The slide containing the cells isincubated, rinsed with PBS and observed under a microscope.

Next, it may be desirable to remove the upper structure of themulti-slide assemblies from the slide plate, typically by inserting asuitable opener to pry the upper structure from the plate. For example,one suitable pry-bar opener is illustrated at 200 at FIG. 1. This openeris illustrated in more detail in FIGS. 14-15. Opener 200 generallyincludes an elongated member 210 having grip ridges 212 andstrengthening ribs 214 disposed on opposing surfaces thereof. A prymember 220, which extends at roughly a 90° angle relative to theelongated member, is levered at point 222 to form a fulcrum from which aseparation force may be applied to a multi-well slide assembly. Member220 typically engages between the upper structure and slide plate of amulti-well slide assembly when the elongated member extends roughlyperpendicular to the plane of the slide plate, with the fulcrum definedon the pry member engaging along the frosted writing surface of theslide-plate.

By virtue of its angled orientation, the opener is able to fit betweenthe various assemblies in the frame to perform the separation. Moreover,when the upper structure of an adjacent assembly in the frame hasalready been removed, the angled orientation of the opener preventsdamage or contamination to the cells on the adjacent slide plate whenremoving the upper structure of an assembly.

Alternatively, the cells may be fixed and processed once the upperstructures have been removed from the slide plates from each assembly.Finally, cover slips may then be applied over the fixed and processedcells, whereby the cells are then ready for visual microscopic analysis.

Cell cultures may be analyzed by various optical viewing devicesincluding microscopes, spectrophotometers, luminometers, etc. Moreover,by virtue of the open access to both surfaces of the slide platesthrough frame 100, analysis may be performed from either side of theslide plates. Moreover, the assemblies may be removed from the frame andseparately analyzed if desired.

Therefore, it should be appreciated that the various embodiments of theinvention provide significant advantages in simplifying and acceleratingdiagnostic testing. Moreover, it has been found that the variousembodiments also provide improved performance over conventional cellculture vessels such as dram vials and culture tubes as they have beenfound to be more sensitive, often experiencing more pronouncedcytopathic effects more quickly. Moreover, it has been found thatcross-contamination is reduced compared to various multi-vesselassemblies such as multi-well plates. Another advantage is that the needfor glass cover slips, as is often required for cell growth and testingwith dram vials or Petri dishes, is eliminated. Moreover, the inherenthazards associated with the use of glass in a laboratory are avoided.Other advantages of the various embodiments of the invention will beapparent to one skilled in the art.

Various modifications may be made to the various embodiments withoutdeparting from the spirit and scope of the invention. Therefore, theinvention lies in the claims hereinafter appended.

1. A method of making a reaction vessel of the type including at leastone well defined by a sidewall member removably coupled to a slideplate, the method comprising: (a) forming a bond between the sidewallmember and the slide plate via ultrasonic welding, the bond sufficientto withstand centrifugation forces; and (b) controllably weakening thebond between the sidewall member and the slide plate via ultrasonicwelding to permit selective separation of the sidewall member from theslide plate.
 2. The method of claim 1, wherein the sidewall memberincludes an energy director disposed along an end thereof, and whereinforming the bond includes welding to a distance slightly less than orsubstantially equal to a length of the energy director.
 3. The method ofclaim 2, wherein the energy director includes a base coupled to asidewall at the end of the sidewall member, the base having a width andthe sidewall having a thickness, wherein weakening the bond includesadditionally welding at least to the remaining length of the energydirector such that the bond has a width that is between the width of thebase of the energy director and the thickness of the sidewall.
 4. Themethod of claim 2, wherein the energy director has a length of about0.019 inches (0.49 mm), and forming the bond includes welding to adistance of about 0.017 inches (0.42 mm), and wherein weakening the bondincludes welding to a distance of about 0.004 inches (0.09 mm).
 5. Themethod of claim 2, wherein forming the bond includes melting the energydirector to a molten state.
 6. The method of claim 1, wherein weakeningthe bond includes welding with a greater pressure than that used duringforming the bond.
 7. The method of claim 6, wherein weakening the bondincludes welding at about 10 psi greater pressure than that used duringforming the bond.
 8. The method of claim 7, wherein weakening the bondincludes welding at about 30 psi, and wherein forming the bond includeswelding at about 20 psi.
 9. The method of claim 1, further comprisingcooling the sidewall member and slide plate prior to forming the bondtherebetween.
 10. The method of claim 1, wherein the sidewall membercomprises an upper structure defining with the slide plate a pluralityof wells.